Synthesis and conformational analysis of efrapeptins.

The efrapeptin family of peptide antibiotics produced by the fungus Tolypocladium niveum, and the neo-efrapeptins from the fungus Geotrichum candidumare inhibitors of F(1)-ATPase with promising antitumor, antimalaria, and insecticidal activity. They are rich in C(α)-dialkyl amino acids (Aib, Iva, Acc) and contain one ß-alanine and several pipecolic acid residues. The C-terminus bears an unusual heterocyclic cationic cap. The efrapeptins C-G and three analogues of efrapeptin C were synthesized using α-azido carboxylic acids as masked amino acid derivatives. All compounds display inhibitory activity toward F(1)-ATPase. The conformation in solution of the peptides was investigated with electronic CD spectroscopy, FT-IR spectroscopy, and VCD spectroscopy. All efrapeptins and most efrapeptin analogues were shown to adopt helical conformations in solution. In the case of efrapeptin C, VCD spectra proved that a 3(10)-helix prevails. In addition, efrapeptin C was conformationally studied in detail with NMR and molecular modeling. Besides NOE distance restraints, residual dipolar couplings (RDC) observed upon partial alignment with stretched PDMS gels were used for the conformational analysis and confirmed the 3(10)-helical conformation.

Chiral, fully extended helical peptides.

The synthesis of the N-protected (blocked) homo-peptide esters from the chiral C(α)-ethyl, C(α)-n-pentylglycine was performed in solution to the hexapeptide level. The conformational propensity exhibited by these oligomers in chloroform solution and in the crystal state was assessed by use of FTIR absorption, NMR, and X-ray diffraction. The results indicated that fully extended helical structures (2.0(5)-helices) are overwhelmingly adopted irrespective of the peptide main-chain length. This oligomeric series is of great interest as it is characterized by the longest C ( i )(α) ,…, C ( i+1 )(α) (per residue) separation achievable in the class of chiral, rigid, helical peptide spacers based on α-amino acids.

Total synthesis, characterization, and conformational analysis of the naturally occurring hexadecapeptide integramide A and a diastereomer.

Integramide A is a 16-amino acid peptide inhibitor of the enzyme HIV-1 integrase. We have recently reported that the absolute stereochemistries of the dipeptide sequence near the C terminus are L-Iva(14)-D-Iva(15). Herein, we describe the syntheses of the natural compound and its D-Iva(14)-L-Iva(15) diastereomer, and the results of their chromatographic/mass spectrometric analyses. We present the conformational analysis of the two compounds and some of their synthetic intermediates of different main-chain length in the crystal state (by X-ray diffraction) and in solvents of different polarities (using circular dichroism, FTIR absorption, and 2D NMR techniques). These data shed light on the mechanism of inhibition of HIV-1 integrase, which is an important target for anti-HIV therapy.

Configurational assignment of D- and L-isovalines in intact, natural, and synthetic peptides by 2D-NMR spectroscopy.

We investigated, by means of 2D-NMR, the naturally occurring and chemically synthesized 16-mer integramides A and B, which belong to a group of bioactive, fungal peptides (peptaibiotics), that are characterized by an abundance of Aib as well as D- and L-Iva residues. The chemical shifts of the C(alpha)-alkyl groups in the Iva enantiomers depend on the alpha-C-atom configuration and on the helical screw sense of the peptides, the latter determined by CD. In the full-length, right-handed helical integramides, as well as in the partial sequences exploited for their total chemical syntheses, the gamma-Me H-atoms of the Et side chain of the D-Iva residues located near the C-terminus are significantly more shielded (delta<0.90 ppm) than those of the L-Iva residues (delta>0.95 ppm). The opposite behavior is observed for the left-handed, synthetic, intermediate Z-Aib-L-Hyp-L-Iva(14)-D-Iva(15)-O(t)Bu. Here, the gamma-Me H-atoms of L-Iva(14) are more shielded (0.838 ppm) than those of D-Iva(15) (0.905 ppm). The chemical-shift difference between the diastereotopic beta-CH(2) H-atoms of the Iva side chains in the right-handed helical peptides is much larger for D-Iva than for L-Iva. For D-Iva(14/15), the values range from 0.38 to 0.63 ppm, whereas, for D-Iva(1), the value is in the range of 0.26-0.31 ppm. In each case, the difference is always larger for the d-Iva than for the l-Iva residues (which is always

Is the backbone conformation of C(alpha)-methyl proline restricted to a single region?

C(alpha)-methyl-L-proline, or L-(alphaMe)Pro, is probably the most conformationally constrained alpha-amino acid. In particular, its omega and phi torsion angles are restricted to about 180 and -60 degrees, respectively, and only three ranges of values are theoretically available for psi in mono- or longer peptides, namely, about -30 degrees (cis', 3(10)/alpha-helical structure), 60 degrees (inverse gamma turn), or 140 degrees (trans', poly(L-Pro)(n) II structure). In this work, we examined the tendency of a number of N(alpha)-acyl dipeptide N'-alkylamides of the type RCO-(alphaMe)Pro-Xxx-NHR' or RCO-Xxx-(alphaMe)Pro-NHR', in which Xxx is L (or D)-Ala, Aib (alpha-aminoisoburyric acid), or L (or D)-(alphaMe)Pro, long enough to fold into intramolecularly hydrogen-bonded gamma or beta turns. The results are compared with those obtained for the corresponding dipeptides based on Pro, a well-known turn-forming residue. For the crystal-state 3D-structural analysis we used X-ray diffraction, whereas our solution conformational analysis was heavily based on the FTIR absorption and (1)H and (13)C NMR spectroscopy techniques. We conclude that (alphaMe)Pro is able to explore both trans' and cis' psi areas of the conformational space, but in (alphaMe)Pro the latter is overwhelmingly more populated, in marked contrast to the Pro preference. This finding is a clear indication that in (alphaMe)Pro the major 3D-structural determinant is the C(alpha)-methyl group. The circular dichroism (CD) signature of a peptide type III' beta-turn conformation is also proposed.

Conformational effects on the electron-transfer efficiency in peptide foldamers based on alpha,alpha-disubstituted glycyl residues.

Peptide foldamers based on alpha,alpha-disubstituted glycyl residues were synthesized and chemically characterized to investigate the effects of the electric field generated by a 3(10)-helix on the rate of intramolecular photoinduced electron-transfer reactions. To this end, two new octapeptides having identical sequences were suitably side-chain functionalized with the same electron-transfer donor-acceptor pair, but inverting the position of the pair along the main chain. The electron-transfer rate constants, measured by time-resolved spectroscopy techniques (nanosecond transient absorption and time-resolved fluorescence), indicated that, in the case of the 3(10)-helix, the electrostatic effect is significant, but smaller than that obtained for alpha-helical peptides. This finding can be likely ascribed to the distortion of the H-bond network with respect to the helical axis taking place in the former secondary structure. Overall, these results could have implications on electron-transfer phenomena in model and biomembranes facilitated by peptaibiotics.

Stereoselective iodocyclization of (S)-allylalanine derivatives: gamma-lactone vs cyclic carbamate formation.

An efficient procedure for highly chemo- and stereoselective cyclization of (S)-allylalanine derivatives is reported (diastereomeric ratios up to 96:4) where the reaction course can be completely controlled by switching from gamma-lactones to cyclic carbamates simply with the proper choice of the amino acid protecting groups. Both processes are stereoconvergent and afford the (S,S)-products in high yields, short reaction times, and mild reaction conditions.

Two-dimensional infrared spectral signatures of 3(10)- and alpha-helical peptides.

Two-dimensional infrared (2D IR) spectra of Calpha-alkylated model octapeptides Z-(Aib)8-OtBu, Z-(Aib)5-L-Leu-(Aib)2-OMe, and Z-[L-(alphaMeVal)]8-OtBu have been measured in the amide I region to acquire 2D spectral signatures characteristic of 3(10)- and alpha-helical conformations. Phase-adjusted 2D absorptive spectra recorded with parallel polarizations are dominated by intense diagonal peaks, whereas 2D rephasing spectra obtained at the double-crossed polarization configuration reveal cross-peak patterns that are essential for structure determination. In CDCl3, all three peptides are of the 3(10)-helix conformation and exhibit a doublet cross-peak pattern. In 1,1,1,3,3,3-hexafluoroisopropanol, Z-[L-(alphaMeVal)]8-OtBu undergoes slow acidolysis and 3(10)-to-alpha-helix transition. In the course of this conformational change, its 2D rephasing spectrum evolves from an elongated doublet, characteristic of a distorted 3(10)-helix, to a multiple-peak pattern, after becoming an alpha-helix. The linear IR and 2D absorptive spectra are much less informative in discerning the structural changes. The experimental spectra are compared to simulations based on a vibrational exciton Hamiltonian model. The through-bond and through-space vibrational couplings are modeled by ab initio coupling maps and transition dipole interactions. The local amide I frequency is evaluated by a new approach that takes into account the effects of hydrogen-bond geometry and sites. The static diagonal and off-diagonal disorders are introduced into the Hamiltonian through statistical models to account for conformational fluctuations and inhomogeneous broadening. The sensitivity of cross-peak patterns to different helical conformations and the chain length dependence of the spectral features for short 3(10)- and alpha-helices are discussed.

Different spectral signatures of octapeptide 3(10)- and alpha-helices revealed by two-dimensional infrared spectroscopy.

Femtosecond two-dimensional infrared (2D IR) spectroscopy is applied to the amide I modes of the terminally protected homo-octapeptide Z-[L-(alphaMe)Val](8)-OtBu in CDCl(3), 2,2,2-trifluoroethanol (TFE), and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) solutions to acquire 2D spectral signatures that distinguish between 3(10)- and alpha-helix structures. Suppression of diagonal peaks by controlling polarizations of IR pulses clearly reveals cross-peak patterns that are crucial for structural determination. A doublet feature is observed when the peptide ester forms a 3(10)-helix in CDCl(3) and TFE and when it is at the initial stage of 3(10)- to alpha-helix transition in HFIP. In contrast, the 2D IR spectrum shows a multiple peak pattern after the peptide ester has acidolyzed and become an alpha-helix in HFIP. Electronic circular dichroism spectra accompanying the acidolysis-driven conformational change are also reported. This is the first report on the experimental 2D IR signature of a 3(10)-helical peptide. These results, using a model octapeptide, demonstrate the powerful capability of 2D IR spectroscopy to discriminate between different helical structures.

Diastereoselective synthesis of (2S,5R)-5-hydroxypipecolic acid and 6-substituted derivatives.

[reaction: see text] Herein, we report a diastereoselective synthesis of the natural product (2S,5R)-5-hydroxypipecolic acid and 6-substituted derivatives thereof. The key step in the synthetic sequence is a novel highly diastereoselective epoxidation reaction of an enantiomerically pure cyclic enamide intermediate.

The Family Approach to the Resolution of Racemates .

The resolution of racemates is revolutionized with the method presented here, in which mixtures ("families") of structurally and stereochemically related resolving agents are used to precipitate salts of acidic or basic racemates rapidly and dependably. The racemate is usually separated in a single operation into enantiomers-the enantiomeric excesses and yields are good to excellent. Reagent mixtures with racemic or achiral components have also been developed.

Triple Hyp→Pro replacement in integramide A, a peptaib inhibitor of HIV-1 integrase: Effect on conformation and bioactivity.

AIDS is produced by HIV-induced infections. HIV integrase is an important enzyme as it is critical for the integration of the HIV genome into that of the host cell. This complex process is exclusively carried out by a viral enzyme not found in the host cell. Therefore, this protein represents a safe target for the development of single or combined anti-HIV therapy. Integramide A is a 16-mer long, effective peptaib inhibitor of HIV-1 integrase. We have previously described a versatile synthetic strategy in solution to afford this natural compound and its diastereomer at positions 14 and 15. We also found that both peptides display a significant inhibitory activity. Here, we present our data on the synthesis in solution, in-depth conformational analysis, and biological activity against HIV-integrase of the analogs of the two above mentioned peptides in which all of the three (2S,4R)-Hyp residues at positions 2, 9, and 13 are replaced by L-Pro. This study definitely confirms that the mixed α-/3(10) - helical conformation of natural integramide A plays a key role in its mechanism of inhibition. Moreover, our data provide evidence that the amphipathic character of this helical structure is not required for the activity of integramide A against HIV-1 integrase. These observations will hopefully help us to further clarify the precise mechanism of inhibition of this interesting peptaib and to identify shorter peptide sequences active against HIV-1 integrase.